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Flow cytometer parameter measurement principle

Flow cytometer is a device for automatic analysis and sorting of cells. It can quickly measure, store and display a series of important biophysical and biochemical characteristic parameters of dispersed cells suspended in liquid, and can sort out specified cell subsets according to preselected parameter ranges. Most flow cytometers are zero-resolution instruments, which can only measure indicators such as total nucleic acid and total protein in a cell, but cannot identify and measure the amount of nucleic acid or protein in a specific part. That is, it has zero detail resolution.

Parameter measurement principle

Flow cytometer can perform multi-parameter measurement at the same time, and the information mainly comes from specific fluorescent signal and non-fluorescent scattering signal. The measurement is carried out in the measurement area, the so-called measurement area is the vertical intersection point of the irradiated laser beam and the liquid flow beam ejected from the orifice. When a single cell in the center of the liquid stream passes through the measurement area, it is irradiated by the laser and scatters light in the entire space with a solid angle of 2π. The wavelength of the scattered light is the same as that of the incident light. The intensity of scattered light and its spatial distribution are closely related to the size, shape, plasma membrane and internal structure of cells, because these biological parameters are also related to the optical properties of cells such as reflection and refraction of light. Unstained cells characteristically scatter light, allowing the analysis and sorting of live unstained cells using the different scattered light signals. Fixed and stained cells, of course, have different scattered light signals than living cells due to changes in optical properties. Scattered light is not only related to the parameters of the cell as the center of scattering, but also to abiotic factors such as the scattering angle and the solid angle at which the scattered light is collected.

In flow cytometry measurements, two kinds of scattered light scattering directions are commonly used: ① forward angle (ie, 0 angle) scatter (FSC); ② side scatter (SSC), also known as 90 angle scatter. The angle mentioned at this time refers to the approximate angle formed between the irradiation direction of the laser beam and the axial direction of the photomultiplier tube that collects scattered light signals. Generally speaking, the intensity of forward angle scattered light is related to the size of the cells, and increases with the increase of the cell cross-sectional area for the same cell population; experiments on spherical live cells show that it is basically the same in the small solid angle range. The size of the cross-sectional area is linear; for cells with complex shapes and orientations, they may vary greatly, and special attention should be paid. The measurement of side scattered light is mainly used to obtain information about the granular nature of the fine structure inside the cell. Although the side scattered light is also related to the shape and size of the cell, it is more sensitive to the refractive index of the cell membrane, cytoplasm and nuclear membrane, and can also give a sensitive response to the larger particles in the cytoplasm.

In actual use, the instrument must first measure the light scattering signal. When light scattering analysis is used in conjunction with fluorescent probes, stained and unstained cells can be identified in a sample. The most efficient use of light scattering measurements is to identify certain subpopulations from a heterogeneous population.

The fluorescence signal mainly includes two parts: ① autofluorescence, that is, the fluorescence emitted by the fluorescent molecules inside the cell after being irradiated with light without fluorescent staining; ② characteristic fluorescence, that is, the fluorescence emitted by the fluorescent dye bound to the cell after the dye is illuminated. Fluorescence, its fluorescence intensity is weak, and the wavelength is also different from that of the irradiated laser. Autofluorescence signals are noise signals that interfere with the resolution and measurement of specific fluorescent signals in most cases. In measurements such as immunocytochemistry, how to improve the signal-to-noise ratio is the key for fluorescent antibodies with low binding levels. Generally speaking, the higher the content of autofluorescent molecules (such as riboflavin, cytochrome, etc.) in cell components, the stronger the autofluorescence; the higher the ratio of dead cells/live cells in the cultured cells, the stronger the autofluorescence ; The higher the proportion of bright cells contained in the cell sample, the stronger the autofluorescence.

The main measures to reduce the interference of autofluorescence and improve the signal-to-noise ratio are: 1. Use brighter fluorescent dyes as much as possible; 2. Choose suitable laser and filter optical systems; 3. Use electronic compensation circuits to compensate for the background contribution of auto-fluorescence.